1. Field of the Invention
The present invention relates to a method for culturing animal, plant and microbial cells effectively.
2. Description of the Prior Art
Recently, production of beneficial substances by culturing animal, plant and microbial cells has been extensively carried out in fields of pharmaceutical, food and chemical industries and the like. In culturing these cells, an effective method for supplying oxygen to a culture medium or a culture broth is important.
Conventionally, in culturing cells, oxygen is supplied by a method in which gas containing oxygen, such as air, is directly introduced into a culture by sparging, namely by a method in which an orifice or a sintered filter is provided at a tip of an air blowing nozzle so that the size of gas bubbles is minimized as much as possible while stirring. Furthermore, a method in which oxygen partial pressure in a gas supplied or inner pressure in a culture device is increased so as to increase a rate of oxygen absorption into a culture has also been developed.
However, in these sparging methods, excessive foaming in a culture causes a problem. In particular, in culturing animal cells, excessive foaming in the culture medium or culture broth is caused by sparging because a culture medium frequently includes serum which contains proteins acting as a surface active agent; as a result, the cells growth is markedly inhibited. Furthermore, in culturing microbial cells, a volume of culture per culture bath has to be reduced because of foaming, which reduce productivity relative to the size of the culture bath. If the forming is excessively vigorous, culturing itself cannot be carried out.
Proposed methods for supplying oxygen more efficiently to cells in order to solve these problems include a method in which an aerobic cultivation of cells is carried out using porous hollow fiber (Japanese Patent Publication No. 7558/1982) and a method in which oxygen is supplied to a culture indirectly via a non-porous separating membrane made of silicone (Japanese Patent Publication No. 20261/1983, Japanese Patent Laid-open No. 309177/1988).
However, the porous membrane as disclosed in Japanese Patent Publication No. 7558/1982 is mechanically strong to some extent, when used for a long period of time, air bubbles may leak from the membrane due to change in blowing pressure, alternatively, membrane becomes hydrophilic due to the presence of surface active substances, thereby allowing the culture to permeate into a gas supplying part.
Furthermore, while the method disclosed in Japanese Patent Publication No. 20261/1983, in which a nonporous separating membrane is used, has an advantage that a culture medium or a culture broth does not permeate into the inside of the hollow fibers; because the oxygen diffusion resistance of the membrane is high, it is necessary to enlarge a membrane area enormously or to use a extremely thin membrane having low diffusion resistance in order to supply sufficient oxygen for the growth of cells. However, such a method which can supply enough oxygen for the cell growth by enlarging the membrane area is not economically advantageous because the amount of the membrane to be used increases excessively.
Furthermore, in the method disclosed in Japanese Patent Laid-open No. 309177/1988, in which a thin silicone-made nonporous separating membrane is used, it is necessary to employ a complicated method of making the silicone-made nonporous separating thin membrane having weak mechanical strength in a form of net, in order to maintain its mechanical strength and to protect the hollow fiber membrane from being torn by unexpected force.
Furthermore, when used as a membrane module, the membrane has to be immobilized at the end using a potting agent; however, adhesion of the nonporous separating membrane to the potting agent is weak because the surface of the membrane is not rough enough. Therefore, the surface of the membrane has to be treated in advance to make the surface rough, which complicates a process for producing the membrane module. Furthermore, when the membrane is practically used in cultivation, the thin membrane which has substantially effective oxygen permeability is mechanically weak and easily smashed by pressure due to cell growth, which results in reduction in gas permeability or destruction of the membrane.